Ohio District's Microbiology Laboratory

Escherichia coli, using the mTEC method

Updated May 2005 

The mTEC agar method is a two-step membrane-filtration method for detection of Escherichia coli (E. coli). This method can be done in the field or laboratory.

THEORY: The first step is incubation of mTEC agar plates at 35°C for 2 hours followed by incubation at 44.5°C for 22-24 hours.  The mTEC agar contains selective and differential agents.  Sodium lauryl sulfate and sodium desoxycholate inhibit Gram + cocci and endospore-forming bacteria.  Brom-cresol purple and brom-phenol red inhibit nontarget bacteria and cause a color change from purple to yellow when lactose is utilized.  Lactose utilization (yellow color) is the basis for presumptive identification of E. coli.

For the second step, after incubation on mTEC, filter membranes containing yellow colonies are transferred to pads saturated with urea-phenol solution.  After 15-20 minutes of incubation on the urea substrate, colonies that are not E. coli turn from yellow to purple.  This color change is caused by the breakdown of urea by the enzyme urease and a subsequent increase in pH.  E. coli is urease negative, and colonies of E. coli remain yellow.

USE:  The mTEC method is recommended for use in monitoring fresh, estuarine, and marine surface waters.  It was specifically developed to be used as a measure of recreational water quality.

MEDIA:  The mTEC medium is available commercially in the dehydrated form from Schleicher & Schuell (800/645-2302, Cat 74217A).  To prepare urea-phenol substrate, ingredients can be purchased from Fisher Scientific (800/766-700): phenol red (Cat AC41724-0050) and urea (Cat U15-500).  See preparation instructions. (Appendix F).

Several investigators, however, have noted problems with the commercial dehydrated media.  The following problems may be related to the shelf life of the dehydrated media:

·         Typical yellow to yellow-brown E. coli colonies turn purple by the end of the 22-24 hour incubation period on mTEC agar.  These colonies remain purple after the urease test.

·         Colonies exhibit confluent growth that is purple in color and spreading in form, covering several grids on a membrane filter.

·         High numbers of variously shaped purple colonies atypical of E. coli are present.

Prepoured plates can be purchased from Schleicher & Schuell (Cat P7300).

Use phosphate buffered dilution water and 0.45 mm membrane filters.  Buffer can be purchased from Hardy Diagnostics (800/346-2766, Cat D699 (99mL) or Cat U193 (500mL)).  See buffer preparation (Appendix M).

REFERENCES: 

U.S. Environmental Protection Agency, 1986.  Test method for Escherichia coli and enterococci in water by the membrane-filter procedure: Cincinnati, Ohio, EPA 600/4-85/076, 24 p.

U.S. Environmental Protection Agency, 2000.  Improved enumeration methods for the recreational water quality indicators: Enterococci and Escherichia coli: Washington, D.C., EPA/821/R-97/004 March 2000.

  The second document can be obtained at  http://www.epa.gov/nerlcwww/index.html

Under E. coli, recreational water methods.

  NWIS PARAMETER CODES:

  31633 E. coli on mTEC agar, colonies per 100 mL